This disclosure relates generally to the field of immunoassays and specifically with immunoassays used to determine concentrations of triiodothyronine (T.sub.3), thyroxine (T.sub.4) and thyroxine binding globulin (TBG) in a fluid sample such as blood serum.
In a radioimmunoassay (RIA) for T.sub.3 or T.sub.4, it is known that the use of blocking agents (sometimes referred to as deblocking agents) improves the assay by either allowing the competitive binding to go faster or by changing the equilibrium point. In general, the function of the blocking agent is to displace the T.sub.3 or T.sub.4 from serum proteins, primarily TBG. Substances that act as blocking agents incude 8-anilino-1-naphthalenesulfonic acid (ANS), merthiolate (thimerosal), dilantin, and many other substances. Such blocking agents and their use in a RIA of T.sub.3 or T.sub.4 are described, for example, in U.S. Pat. No. 3,911,096 to I. J. Chopra, the teachings of which are incorporated herein by reference.
Because one of the essential steps in most RIA's involves the separation of labeled immunochemically complexed products from radio labelled substances which are not so complexed, attention has been drawn recently to the use of solid phase techniques in RIA. Solid phase RIA (SPRIA) involves the use of an insoluble carrier to which antibodies or antigenic substances can be attached in an active form. Since the solid phase is insoluble, the RIA separation step is facilitated. Examples of the various carrier materials which can be used in SPRIA can be founrd in U.S. Pat. No. 3,555,143 to Axen et al. (organic carriers) and U.S. Pat. No. 3,652,761 to Weetall (inorganic carriers).
In preliminary investigations of the reaction kinetics of a typical SPRIA using an immobilized antibody (IMA) to thyroxine, we found that the complexation rate was definable as: EQU Rate = [FT.sub.4 ][IMA],
where [FT.sub.4 ] represents the free T.sub.4 concentration. By combining this observation with a further observation that blocking agents in T.sub.4 assays serve primarily to displace T.sub.4 from TBG (an .alpha.-globulin), we were led to the discovery that TBG concentrations could be determined by a novel method, the details of which are described herein.